Application of brutinib and combined pharmaceutical composition thereof in treatment of leukemia

Abstract

The invention discloses application of brutinib and a combined pharmaceutical composition thereof in treatment of leukemia, and the leukemia comprises acute myelogenous leukemia, chronic myelogenous leukemia, T acute lymphoblastic leukemia and B acute lymphoblastic leukemia. The invention creatively finds that the brutinib has a remarkable synergistic effect on treatment of the leukemia when being combined with the vinca, the cytarabine or the daunorubicin for use, and a brand new drug combination scheme provided by the invention provides a more effective drug combination choice for treatment of the leukemia, and is expected to improve the treatment effect of patients with the leukemia.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of brigatinib and its combination drug compositions in the treatment of leukemia. Background Technology

[0002] Leukemia is a common hematologic malignancy, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), T-acute lymphoblastic leukemia (T-ALL), and B-acute lymphoblastic leukemia (BALL). Although chemotherapy, targeted therapy, immunotherapy, and bone marrow transplantation have brought these leukemias under some control, leukemia patients are highly prone to relapse, and after relapse, they lose sensitivity to chemotherapy drugs. Patients often die from drug resistance and various complications, which remains a significant challenge in leukemia treatment.

[0003] Cytarabine, daunorubicin, and venetoclax (ABT-199) are common drugs for treating leukemia. Despite exhibiting good clinical anti-leukemic activity and significantly improving treatment outcomes for some leukemia patients, drug resistance, prevalent in relapsed or refractory leukemia patients, remains a key bottleneck limiting treatment efficacy, ultimately leading to treatment failure and death risk for some AML patients. Therefore, developing novel treatment regimens that can reverse drug resistance or enhance their anti-leukemic activity is of crucial clinical significance and research value for overcoming current treatment challenges and further improving the overall cure rate of leukemia.

[0004] Brigatinib is a second-generation oral tyrosine kinase inhibitor that has been approved by the US FDA, European EMA, and China's National Medical Products Administration (NMPA) for the treatment of ALK-positive non-small cell lung cancer (NSCLC). However, to date, there are no studies or reports on the use of brigatinib alone or in combination with verneclax, cytarabine, or daunorubicin for the treatment of leukemia. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems existing in the field, the purpose of the present invention is to provide the application of brigatinib and its combination drug composition in the treatment of leukemia.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution: The first aspect of the invention provides the use of brigatinib or in combination with verneclax, cytarabine or daunorubicin in the preparation of a medicament for the prevention and / or treatment of leukemia.

[0007] Furthermore, the application includes the use of any of the following drugs in the preparation of medicaments for the prevention and / or treatment of leukemia: (1) Brigatinib or its pharmaceutically acceptable salts; (2) Brigatinib or its pharmaceutically acceptable salts in combination with Venecella or its pharmaceutically acceptable salts; (3) Brigatinib or its pharmaceutically acceptable salts in combination with cytarabine or its pharmaceutically acceptable salts; (4) Brigatinib or its pharmaceutically acceptable salts in combination with daunorubicin or its pharmaceutically acceptable salts; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to verneclax or its pharmaceutically acceptable salt is 1:1 to 1:8; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to cytarabine or its pharmaceutically acceptable salt is 1:0.1-1:5; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to daunorubicin or its pharmaceutically acceptable salt is 25:1-100:1; Optionally, the leukemia is acute myeloid leukemia, chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, or B-cell acute lymphoblastic leukemia.

[0008] In this invention, brigatinib, an anaplastic lymphoma kinase (ALK) inhibitor, is approved for the treatment of ALK-positive advanced or metastatic non-small cell lung cancer. Brigatinib effectively inhibits ALK and ALK fusion proteins, and also exhibits potent inhibition of target molecules such as ROS1, FLT3, and EGFR mutants, demonstrating good therapeutic effects in both in vivo and in vitro experiments. This invention does not impose any particular restrictions on the specific source of brigatinib; those skilled in the art can obtain it through conventional channels.

[0009] In this invention, Venetoclax is a novel selective Bcl-2 inhibitor primarily used for the treatment of specific types of leukemia, such as chronic lymphocytic leukemia (CLL) and AML. Its mechanism of action involves inhibiting the BCL-2 protein (an anti-apoptotic protein), thereby inducing apoptosis in cancer cells and controlling disease progression. This invention does not impose any particular limitation on the specific source of Venetoclax; those skilled in the art can obtain it through conventional means.

[0010] In this invention, cytarabine is an antimetabolite chemotherapy drug, belonging to the class of antitumor drugs, mainly used to treat hematologic malignancies such as acute leukemia and lymphoma. Its mechanism of action involves competitively inhibiting DNA polymerase through its metabolite Ara-CTP, blocking DNA synthesis, and inhibiting tumor growth and proliferation. It is one of the fundamental drugs in clinical chemotherapy regimens. This invention does not impose any particular restrictions on the specific source of cytarabine; those skilled in the art can obtain it through conventional channels.

[0011] In this invention, daunorubicin is a first-generation anthracycline antitumor antibiotic, originally extracted from *Streptomyces peucetius*. This drug is primarily used to treat acute leukemia (granulocytic, lymphocytic, monocytic, granulocytomonocytic), erythroleukemia, chronic myeloid leukemia, and malignant lymphoma, and has been extended to neuroblastoma. This invention does not impose any particular restriction on the specific source of the daunorubicin; those skilled in the art can obtain it through conventional means.

[0012] In this invention, the pharmaceutically acceptable salt refers to a salt that is pharmaceutically acceptable. It refers to the salt form generated by the reaction of a drug active ingredient (such as brigatinib, veneclade, cytarabine, daunorubicin) with a suitable acid or base. Its core characteristics are that it is pharmaceutically safe and non-toxic, and can maintain the therapeutic effect of the original drug active ingredient. At the same time, it can improve the physicochemical properties (such as solubility, stability, bioavailability) or administration convenience of the original drug, and is suitable for the preparation of various dosage forms such as oral preparations and injections.

[0013] Specifically, pharmaceutically acceptable salts do not damage the molecular structure or mechanism of action of the original drug's active ingredients, maintaining its core efficacy of inhibiting leukemia cell proliferation and promoting apoptosis. This ensures that the synergistic effect (e.g., a CI < 1) of brigatinib monotherapy or its combination with verneclade, cytarabine, or daunorubicin remains unaffected. Furthermore, the resulting salts meet pharmaceutical standards, do not produce toxic or harmful metabolites after entering the human body, and do not cause additional toxic side effects, ensuring safety and harmlessness for leukemia patients at clinical therapeutic doses. They can also specifically address potential deficiencies of the original drug to adapt to the needs of various formulation types, such as improving solubility, enhancing stability, and improving bioavailability.

[0014] In some embodiments, the pharmaceutically acceptable salt is an acid addition salt. If the drug molecule contains basic groups such as amino or guanidine groups (e.g., brigatinib, veneclade, daunorubicin), it can react with inorganic or organic acids to form an acid addition salt. The inorganic acid salts include, but are not limited to, hydrochlorides, sulfates, phosphates, and nitrates. For example, brigatinib can react with hydrochloric acid to form brigatinib hydrochloride, which may have better water solubility than the free base form, making it more suitable for preparing injections or oral suspensions. The organic acid salts include, but are not limited to, mesylates, ethanesulfonates, citrates, tartrates, maleates, and fumarates. These salts often have better biocompatibility, and some can also reduce the drug's irritation to the gastrointestinal tract, meeting the taste and safety requirements of the oral formulations (e.g., syrups, granules).

[0015] In some embodiments, the pharmaceutically acceptable salt is a base addition salt. If the drug molecule contains acidic groups such as carboxyl or sulfonic acid groups (e.g., cytarabine), it can react with inorganic or organic bases to form a base addition salt. The inorganic base salt includes, but is not limited to, sodium, potassium, calcium, and magnesium salts. For example, cytarabine can be formulated as cytarabine sodium salt, which has excellent water solubility, meeting the requirements for rapid dissolution and precise administration of injectable preparations (e.g., intravenous infusion solutions), and is suitable for high-dose chemotherapy in the acute phase of leukemia. The organic base salt includes, but is not limited to, ammonium, ethanolamine, and diethanolamine salts. These salts are mostly suitable for oral formulations and can further improve the solubility and stability of the drug.

[0016] In a specific embodiment of the present invention, the synergistic effect of the combination of brigatinib and veneclade, brigatinib and cytarabine, and brigatinib and daunorubicin was further verified by the combination drug CI index. The combination drug CI index is a method for evaluating the combined effect of drugs. This method is based on the dose-response curve of the drugs and calculates the ratio of the actual effect to the theoretical effect when using combination drugs (the specific calculation method is the well-known method of calculating the combination drug index CI using CalcuSyn software. CalcuSyn software calculates the CI index based on the Chou-Talalay theory by fitting the dose-response curves of the single drug and the combination group). A CI value less than 1 indicates a synergistic effect, equal to 1 indicates an additive effect, and greater than 1 indicates an antagonistic effect.

[0017] In specific embodiments of the present invention, experiments have demonstrated the effectiveness of brigatinib and veneclade (concentration ratios of 0.625 μM:1.25 μM (mass ratio 1:3.20), 0.625 μM:2.5 μM (mass ratio 1:6.40), 1.25 μM:1.25 μM (mass ratio 1:1.60), 1.25 μM:2.5 μM (mass ratio 1:3.20)), and brigatinib and cytarabine (concentration ratios of 0.625 μM:0.625 μM (mass ratio 1:0.45), 0.625 μM:1.25 μM (mass ratio 1:0.896), 1.25 μM:0.625 μM (mass ratio 1:0.224), 1.25 μM:1.25 μM:1.25 μM). The combined use of brigatinib and daunorubicin (concentration ratios of 0.625 μM:0.0125 μM (mass ratio 1:0.0194), 0.625 μM:0.025 μM (mass ratio 1:0.0388), 1.25 μM:0.0125 μM (mass ratio 1:0.0097), and 1.25 μM:0.025 μM (mass ratio 1:0.0194)) showed significant synergistic effects (CI<1) in the treatment of leukemia.

[0018] Furthermore, the brigatinib or its combination with verneclade, cytarabine or daunorubicin significantly inhibits the proliferation of leukemia cells and / or promotes apoptosis of leukemia cells.

[0019] Furthermore, the drug comprises an effective amount of brigatinib, an effective amount of brigatinib and veneclade, an effective amount of brigatinib and cytarabine, or an effective amount of brigatinib and daunorubicin.

[0020] Furthermore, the drug also contains pharmaceutically acceptable excipients; Optionally, the pharmaceutically acceptable excipients include diluents, binders, disintegrants, lubricants, suspending agents, stabilizers, preservatives, and / or osmotic pressure regulators; Optionally, the dosage form of the drug is an oral preparation, an injection, or an intravenous infusion; Optionally, the oral preparation is a tablet, capsule, granule, suspension, powder, pill, emulsion or syrup; Optionally, the injectable is a lyophilized powder for injection, an injection solution, an injection suspension, an injection emulsion, or a liposome injection.

[0021] In some implementations, the diluent is also called a filler. Its core function is to increase the volume and weight of the drug formulation when the content of the active ingredient is low (such as when the proportion of a certain component in a braganaib monotherapy or combination formulation is small), so that the formulation meets the specifications required for production, packaging, and clinical use, while ensuring the stability of the formulation form and accurate dosage. In oral formulations (such as tablets and capsules), lactose is a widely used diluent. It has good flowability and compressibility, is compatible with a variety of drug components, and has a good taste, making it suitable for braganaib tablets or capsules that need to be taken orally. Microcrystalline cellulose is also commonly used. It can not only be used as a diluent, but also has certain binding and disintegrant properties, which helps to improve the hardness and disintegration performance of tablets, especially suitable for combination drug tablets with high requirements for the physical properties of the formulation. Starch (such as corn starch and potato starch) is also a traditional diluent. It is inexpensive, but its flowability is relatively poor. It is often used in combination with other excipients. For example, in braganaib granules, it can be mixed with lactose as a filler base.

[0022] In some embodiments, the adhesive enables drug powders or granules to adhere to each other, forming clumps or granules with a certain strength and shape. This ensures that the formulation (especially the contents of tablets and capsules) is not easily broken during production, transportation, and storage, while also guaranteeing the hardness and abrasion resistance of the tablets to meet the process requirements of subsequent tableting or capsule filling. Povidone (PVP) is a water-soluble adhesive that dissolves in water or ethanol solutions. It forms viscous clumps by encapsulating drug particles and is suitable for the preparation of brigatinib combination formulations where water solubility is not a particular concern. Furthermore, its adhesive strength can be flexibly controlled by adjusting the concentration. Hydroxypropyl methylcellulose (HPMC) has good film-forming and adhesive properties. When used in tablets, it enhances the adhesion of granules and forms a protective film on the tablet surface, reducing the drug's contact with the external environment and improving stability. It is suitable for oral tablets combining brigatinib and cytarabine. Starch paste (such as 10% corn starch paste) is a traditional water-based adhesive that is low in cost and high in safety. It is often used in formulations where moderate adhesive strength is required, such as the preparation of ordinary brigatinib tablets.

[0023] In some implementations, the main function of the disintegrant is to rapidly disintegrate oral formulations (such as tablets and capsules) into fine particles or powder after entering the gastrointestinal tract, increasing the contact area between the drug and gastrointestinal digestive fluids, accelerating the dissolution and release of the active pharmaceutical ingredient, and ensuring that the drug can be absorbed by the body in a timely manner and exert its efficacy. Cross-linked povidone (PVPP) has extremely strong water absorption and swelling properties, expanding its volume dozens of times after absorbing water. It can rapidly disrupt the structure of tablets, promoting their disintegration, and is unaffected by pH value. It is suitable for oral tablets combining brigatinib and daunorubicin, ensuring effective disintegration under different gastrointestinal environments. Sodium carboxymethyl starch (CMS-Na) is a highly efficient disintegrant with a high water absorption and swelling rate, rapid disintegration speed, and no adverse effects on drug stability. It is often used in formulations requiring high disintegration speed, such as brigatinib immediate-release tablets. Low-substituted hydroxypropyl cellulose (L-HPC) generates a certain swelling force and capillary action after absorbing water, promoting tablet disintegration and improving particle flowability. It is suitable for synergistic use with other excipients to improve the overall performance of the formulation.

[0024] In some implementations, the lubricant is primarily used to reduce friction between drug powder or granules during production (e.g., between granules and the die during tableting, or between granules and the capsule shell during capsule filling), preventing material from sticking to equipment surfaces, ensuring smooth production, and improving tablet appearance (e.g., reducing spots and roughness on tablet surfaces), thus enhancing the quality stability of the formulation. Magnesium stearate is the most commonly used lubricant, possessing excellent lubrication properties. It effectively reduces the coefficient of friction between granules and the die, improves tableting efficiency, and is used in small quantities (typically 0.1%-1%), having minimal impact on drug stability and disintegration. It is widely used in the preparation of various oral tablets of brigatinib. Micronized silica (silica) has excellent flowability and anti-sticking properties and is often used as an auxiliary component of lubricants. When used in combination with magnesium stearate, it can further improve granule flowability and reduce material sticking to the wall, especially suitable for brigatinib combination formulations with poor flowability. Talc also has some lubrication and flowability effects, but its lubrication effect is relatively weak. It is often used in formulations requiring high lubricant tolerance or improved tablet gloss.

[0025] In some implementations, the suspending agent is mainly used in suspensions (such as oral suspensions and injectable suspensions) to increase the viscosity of the dispersion medium, reduce the sedimentation rate of drug particles, and make the drug particles uniformly dispersed in the medium to form a stable suspension system. This avoids uneven concentration of drug particles due to sedimentation, ensures accurate dosage each time it is taken or injected, and reduces the irritation of drug particles to the gastrointestinal tract or injection site. Hydroxypropyl methylcellulose (HPMC) is widely used in oral suspensions. It forms a viscous colloidal solution, effectively hindering drug particle sedimentation, and has good biocompatibility with minimal gastrointestinal irritation. It is suitable for the preparation of brigatinib oral suspensions, making it convenient for children, the elderly, and other patients with swallowing difficulties. Gum arabic is a natural suspending agent with good emulsifying and suspending properties. It can form a stable suspension system with drug particles and has high safety, and is often used in traditional oral suspensions. In injectable suspensions, polysorbate 80 (Tween 80) can not only act as a suspending agent but also play a certain role in solubilization, helping to improve the stability of drug particles and reduce irritation during injection. It can be used in the preparation of brigatinib injectable suspensions.

[0026] In some implementations, the stabilizer is used to prevent the active ingredients in the drug formulation (such as brigatinib, veneclade, etc.) from undergoing degradation, oxidation, hydrolysis or polymerization reactions due to factors such as light, temperature, humidity, and oxygen during production, storage and transportation, so as to ensure the chemical stability and biological activity of the drug and ensure that the efficacy and quality of the formulation remain unchanged within its shelf life. Antioxidants prevent drug oxidative degradation. For example, vitamin C (ascorbic acid) and vitamin E (tocopherol) are often used in preparations containing easily oxidized components. If a component in a brigatinib combination preparation is easily oxidized, an appropriate amount of antioxidant can be added. Metal chelators (such as disodium edetate, EDTA-2Na) can bind to trace metal ions that may be present in the preparation (such as iron and copper ions introduced during the production process), preventing metal ions from catalyzing drug degradation reactions. They are suitable for drug preparations that are sensitive to metal ions. pH adjusters (such as hydrochloric acid, sodium hydroxide, and phosphate buffer) can adjust the pH value of the preparation to keep the drug in a stable pH environment and reduce drug hydrolysis caused by pH changes. For example, phosphate buffer is often added to brigatinib injections to maintain the pH stability of the preparation.

[0027] In some implementations, the preservative is mainly used in preparations containing water or other liquid components (such as oral suspensions, syrups, and diluents of injections) to inhibit the growth and reproduction of microorganisms (such as bacteria, fungi, and yeasts), prevent the preparation from being contaminated by microorganisms during production, storage, and use, and avoid the deterioration of the preparation, reduction of efficacy, or even the risk of medication safety (such as infection) caused by microbial contamination. Sodium benzoate is a commonly used preservative in oral preparations. It has good antibacterial effects in acidic environments and can effectively inhibit the growth of bacteria and fungi. It is often used in brigatinib syrup or oral suspensions and has a high safety profile, with no adverse effects on the human body when used within the prescribed dosage. Potassium sorbate has a broad antibacterial spectrum, inhibiting bacteria, fungi, and yeasts. It is also stable and unaffected by pH (effective within a pH range of 3-6), making it suitable for various oral liquid preparations. In sterile preparations such as injections or ophthalmic preparations, benzalkonium chloride is commonly used as a preservative. It has strong antibacterial effects, rapid onset of action, and low irritation to the human body at the prescribed concentration (usually 0.01%-0.1%). It can be used in the preparation of brigatinib injections (if it is a multi-dose injection).

[0028] In some implementations, the osmolarity regulator is mainly used in injectable and ophthalmic preparations that directly enter the cavities or circulatory system of the body. By adjusting the osmolarity of the preparation, it makes it equal to or close to the osmolarity of human body fluids (such as blood and tears) (isotonic or slightly hypertonic), avoiding cell dehydration, shrinkage, swelling, or rupture caused by osmolarity differences, reducing irritation to the injection site, and improving medication safety and comfort. Sodium chloride is the most commonly used osmolarity regulator. By adjusting its concentration, the injection can be made isotonic (e.g., 0.9% sodium chloride solution is an isotonic solution), which is suitable for the preparation of brigatinib injection, ensuring that the preparation will not cause osmolarity disturbances after entering the bloodstream. Glucose can also be used as an osmolarity regulator, with a commonly used concentration of 5%-10%. It not only regulates osmolarity but also provides energy to the body, making it suitable for patients who need energy supplementation or for brigatinib injection preparations compatible with glucose injection. Glycerin has a certain osmolarity regulating effect and can also play a role in moisturizing and suspending, improving medication comfort.

[0029] In some implementations, the oral formulation is one of the most widely used dosage forms in clinical practice, offering advantages such as convenient administration, high patient compliance, and no need for professional medical personnel to administer it. It is suitable for long-term maintenance therapy or treatment during the stable phase of leukemia. In this invention, oral formulations of brigatinib monotherapy or in combination with verneclade, cytarabine, or daunorubicin include, but are not limited to, tablets, capsules, granules, suspensions, powders, pills, emulsions, or syrups.

[0030] The tablets are solid dosage forms made by mixing the active pharmaceutical ingredient with excipients such as diluents, binders, disintegrants, and lubricants, followed by granulation and tableting. They are characterized by accurate dosage, high stability, and ease of carrying and storage.

[0031] The capsule is a preparation in which drug powder, granules or pellets are filled into a hard capsule shell or made into a soft capsule. It has the advantages of masking unpleasant drug odors, improving drug stability and achieving targeted release.

[0032] The granules are dry granular preparations with a certain particle size, made by mixing the drug with suitable excipients. They can be swallowed directly or dissolved in an appropriate amount of water before being taken.

[0033] The suspension is a liquid suspension preparation that can be taken orally, made by mixing a poorly soluble drug with suspending agents, stabilizers, preservatives and other excipients. The drug is dispersed in the liquid medium in the form of microparticles.

[0034] The powder is a powdered preparation made by pulverizing and mixing the drug with suitable excipients. It has the characteristics of simple preparation process, high drug dispersibility and fast absorption speed.

[0035] The pills are spherical or near-spherical solid preparations made by mixing drugs with suitable excipients. Traditional pills (such as honey pills and water pills) have the characteristics of mild and long-lasting effects, while modern pills (such as drop pills) can achieve rapid onset of action.

[0036] The emulsion is a liquid preparation in which a drug (oil-soluble or water-soluble) is dispersed in an immiscible liquid medium and formed with the help of an emulsifier. It is divided into oil-in-water (O / W) and water-in-oil (W / O) types.

[0037] The syrup is a liquid preparation made by dissolving or suspending the drug in a concentrated sucrose aqueous solution. It often contains preservatives, sweeteners, flavorings and other excipients, and has the characteristics of good taste and high patient compliance.

[0038] In some embodiments, the injectable is a sterile preparation made by combining a drug with suitable excipients for injection into the body, capable of directly entering the bloodstream or interstitial space to rapidly exert its therapeutic effect. It is suitable for patients in the acute phase of leukemia, those with severe conditions, or those who do not absorb oral medications well. The injectables in this invention include, but are not limited to: lyophilized powder injections, injection solutions, injection suspensions, injection emulsions, or liposome injections.

[0039] The lyophilized powder injection is a sterile powder preparation made by freeze-drying a drug solution. It needs to be dissolved in a suitable solvent (such as physiological saline or water for injection) before injection.

[0040] The injection solution is a sterile, clear liquid preparation made by dissolving or dispersing the drug in a suitable solvent, which can be directly used for injection (such as intravenous injection, intramuscular injection, or subcutaneous injection).

[0041] The injectable suspension is a sterile liquid suspension preparation that can be injected, made by mixing poorly soluble drugs with suspending agents, stabilizers and other excipients. The drug is dispersed in the liquid medium in the form of microparticles.

[0042] The injectable emulsion is a sterile emulsion liquid preparation in which a drug (oil-soluble or lipid-soluble) is dispersed in an aqueous phase with the help of an emulsifier. It is mainly an oil-in-water (O / W) emulsion and can be administered by intravenous injection.

[0043] The liposome injection is a sterile injectable preparation made by encapsulating drugs in vesicles formed by a lipid bilayer, and has the characteristics of targeting, sustained release, and reduced drug toxicity.

[0044] In some implementations, the intravenous infusion is a preparation that slowly delivers the drug into the body via intravenous infusion. Its administration rate is controllable, maintaining a stable blood drug concentration. It is suitable for treatment scenarios involving high-dose chemotherapy for leukemia or requiring precise control of the drug delivery rate. It is primarily used in the induction remission phase of leukemia, when the patient's condition is severe and requires rapid, large-volume drug administration to control disease progression. The intravenous infusion is typically a solution of the drug as an injection or a lyophilized powder for injection, and must be prepared strictly according to aseptic techniques to avoid contamination. Furthermore, a precision infusion set must be used during the infusion process to ensure accurate infusion rate, maintain a stable blood drug concentration, and guarantee the synergistic effect of brigatinib with combination therapies.

[0045] A second aspect of the invention provides a pharmaceutical composition for the prevention and / or treatment of leukemia.

[0046] Furthermore, the pharmaceutical composition comprises any one of the following drugs: (1) Brigatinib or its pharmaceutically acceptable salts; (2) Brigatinib or its pharmaceutically acceptable salts in combination with Venecella or its pharmaceutically acceptable salts; (3) Brigatinib or its pharmaceutically acceptable salts in combination with cytarabine or its pharmaceutically acceptable salts; (4) Brigatinib or its pharmaceutically acceptable salts in combination with daunorubicin or its pharmaceutically acceptable salts; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to verneclax or its pharmaceutically acceptable salt is 1:1 to 1:8; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to cytarabine or its pharmaceutically acceptable salt is 1:0.1-1:5; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to daunorubicin or its pharmaceutically acceptable salt is 25:1-100:1.

[0047] A third aspect of the present invention provides a pharmaceutical preparation for the prevention and / or treatment of leukemia.

[0048] Furthermore, the pharmaceutical preparation comprises the pharmaceutical composition described in the second aspect of the present invention.

[0049] Furthermore, the dosage form of the pharmaceutical preparation is an oral preparation, an injection, or an intravenous infusion; Optionally, the oral preparation is a tablet, capsule, granule, suspension, powder, pill, emulsion or syrup; Optionally, the injectable is a lyophilized powder for injection, an injection solution, an injection suspension, an injection emulsion, or a liposome injection.

[0050] A fourth aspect of the present invention provides a method for inhibiting the proliferation of leukemia cells and / or promoting apoptosis of leukemia cells in vitro for non-therapeutic purposes.

[0051] Furthermore, the method includes treating leukemia cells with any of the following drugs: (1) Brigatinib or its pharmaceutically acceptable salts; (2) Brigatinib or its pharmaceutically acceptable salts in combination with Venecella or its pharmaceutically acceptable salts; (3) Brigatinib or its pharmaceutically acceptable salts in combination with cytarabine or its pharmaceutically acceptable salts; (4) Brigatinib or its pharmaceutically acceptable salts in combination with daunorubicin or its pharmaceutically acceptable salts; Optionally, the leukemia cells are Kasumi-1, SKNO-1, MOLM-13, NOMO-1, HL-60, THP-1, MV4-11, NB4, U937, OCI-AML5, CCRF-CEM, HPB, KU812, or Sup-B15.

[0052] Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to verneclax or its pharmaceutically acceptable salt is 1:1 to 1:8; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to cytarabine or its pharmaceutically acceptable salt is 1:0.1-1:5; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to daunorubicin or its pharmaceutically acceptable salt is 25:1-100:1; Optionally, the leukemia is acute myeloid leukemia, chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, or B-cell acute lymphoblastic leukemia.

[0053] The fifth aspect of the invention provides for any of the following applications: (1) The use of brigatinib or in combination with verneclax, cytarabine or daunorubicin in the preparation of pharmaceutical formulations for the prevention and / or treatment of leukemia; (2) The use of brigatinib or in combination with verneclax, cytarabine or daunorubicin in the preparation of drugs for the purpose of inhibiting the proliferation of leukemia cells and / or promoting the apoptosis of leukemia cells in vitro for non-therapeutic purposes; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to verneclax or its pharmaceutically acceptable salt is 1:1 to 1:8; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to cytarabine or its pharmaceutically acceptable salt is 1:0.1-1:5; Optionally, the mass ratio of brigatinib or its pharmaceutically acceptable salt to daunorubicin or its pharmaceutically acceptable salt is 25:1-100:1; Optionally, the leukemia is acute myeloid leukemia, chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, or B-cell acute lymphoblastic leukemia.

[0054] Furthermore, the present invention also provides a method for preventing and / or treating leukemia, the method comprising: administering to a subject in need a preventive and / or therapeutically effective amount of the drug, pharmaceutical composition and / or pharmaceutical preparation as described above; Optionally, the leukemia is acute myeloid leukemia, chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, or B-cell acute lymphoblastic leukemia.

[0055] In some embodiments, the effective amount refers to the amount of the drug, pharmaceutical composition, or pharmaceutical preparation of the present invention, as described above, sufficient to produce the desired activity when administered to a subject with the need. When a combination of active ingredients is administered, the effective amount of the combination may or may not include the amounts of each ingredient that might be effective when administered alone. The exact amount required will vary from subject to subject, depending on the subject's species, age and general condition, the severity of the condition being treated, the specific type of drug used, the method of administration, etc.

[0056] In some implementations, the routes of administration include, but are not limited to: subcutaneous, intravenous, oral, intra-arterial, parenteral, intranasal, intracardiac, rectal, intramuscular, inhalation, intraperitoneal, intradermal, percutaneous, or otherwise, such as by implantation.

[0057] In some implementations, the prevention refers to reducing the probability of healthy individuals or high-risk individuals developing leukemia, or delaying the progression of the disease, by applying the drugs, pharmaceutical compositions, or pharmaceutical preparations described above. The core objective is to block the development of leukemia "from nothing to something" or "from latent to overt." The target population for the prevention described in this invention mainly includes two categories: high-risk individuals for leukemia and individuals who have achieved remission from leukemia.

[0058] In some implementations, the treatment refers to the administration of the drugs, drug compositions, or drug preparations of the present invention to patients diagnosed with leukemia to achieve the goals of controlling disease progression, alleviating symptoms, and improving prognosis. The core is to intervene in AML that has already occurred. Specifically, the treatment goals can be divided into the following three categories: induction therapy (suitable for newly diagnosed or relapsed AML patients, especially those with rapid disease progression and a high proportion of bone marrow blast cells), consolidation therapy (suitable for AML patients after induction therapy, with the aim of further eliminating residual leukemia cells (minimal residual disease) in the body and reducing the risk of relapse), and palliative therapy (suitable for older AML patients (e.g., over 75 years old), those with poor physical condition (e.g., multiple organ dysfunction), or those who are intolerant to conventional high-intensity chemotherapy).

[0059] In some implementations, the subjects include various animal groups, including both human individuals and non-human animals. The specific types of non-human animals can be divided into vertebrates and non-vertebrates: Vertebrates include mammals and non-mammals. Mammals include non-human primates (especially higher primates closely related to humans), livestock such as sheep, pigs, and cattle, pets such as dogs and cats, and rodents commonly used in experimental research (such as mice and rats), guinea pigs, rabbits, and other mammals such as goats; non-mammals include birds such as chickens, amphibians (such as frogs and toads), reptiles (such as lizards and snakes), etc. In specific implementations of this invention, from the perspective of the targeted and practical application of the drug, the subjects are preferably humans.

[0060] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention groundbreakingly discovers that brigatinib, when used in combination with verneclade, cytarabine, or daunorubicin, exhibits a significant synergistic effect (CI<1) in the treatment of leukemia. Its inhibitory effect on the proliferation of leukemia cells (acute myeloid leukemia cells, chronic myeloid leukemia cells, T-cell acute lymphoblastic leukemia cells, or B-cell acute lymphoblastic leukemia cells) is significantly superior to that of single-agent therapy. The novel combination therapy provided by this invention offers a more effective drug combination option for the treatment of leukemia, and is expected to improve the treatment outcomes for leukemia patients. Furthermore, this invention lays the foundation for further in-depth exploration of the molecular mechanisms of drug combination effects and the conduct of more related clinical studies, opening up new avenues for the treatment of leukemia. Attached Figure Description

[0061] Figure 1AML cell line MOLM-13 was treated with different concentrations of the ALK inhibitor brigatinib and venetoclax, cytarabine, and daunorubicin, alone or in combination, for 48 hours; T-ALL cell line CCRF-CEM, B-ALL cell line Sup-B15, and CML cell line KU812 were treated with different concentrations of the ALK inhibitor brigatinib alone for 48 hours. h. Cell death was detected by Annexin V-FITC / PI double staining and flow cytometry. The synergy coefficient (CI, CI<1 indicates that the two drugs have a synergistic effect) was calculated using Calcusyn software. Figure A: Representative flow cytometry plot; Figure B: Flow cytometry apoptosis statistics, **** represents p<0.0001, *** represents p<0.001; Figure C: Representative flow cytometry plot and flow cytometry apoptosis statistics, **** represents p<0.0001, ** represents p<0.001. Figure 2 Leukemia cell lines Kasumi-1, SKNO-1, MOLM-13, NOMO-1, HL-60, THP-1, MV4-11, NB4, U937, OCI-AML5, CCRF-CEM, HPB, KU812, or Sup-B15 were treated with different concentrations of the ALK-selective inhibitor Brigatinib for 72 h. The viable cell percentage was detected using the CCK-8 assay, and the IC50 was calculated using GraphPad Prism. 50 value. Detailed Implementation

[0062] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0063] Example 1: Brigatinib alone or in combination with other drugs for the synergistic treatment of leukemia 1. Experimental materials Leukemia cell lines (MOLM-13, CCRF-CEM, HPB, KU812, Sup-B15), AML clinical sample cells.

[0064] 2. Experimental Methods Annexin V / PI staining and flow cytometry were used to detect cell apoptosis. The specific experimental steps are as follows: Leukemia cells (2×10) 5 After 48 h of treatment with the drug per well, cells were collected, centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was washed with 1 mL of pre-chilled PBS and transferred to a 1.5 mL EP tube. The cells were centrifuged at 2000 rpm for 5 minutes, and the supernatant was discarded as much as possible. 50 μL of binding buffer (1× Binding Buffer) was added to the 1.5 mL EP tube, followed by 0.5 μL of Annexin-FITC, and the mixture was incubated at room temperature in the dark for 15 minutes. 0.5 μL of propargite iodide (PI) was added and the mixture was incubated at room temperature in the dark for 5 minutes. 200 μL of binding buffer was added to stop the reaction and the mixture was incubated. Data was analyzed by flow cytometry within 1 hour, and the data files were saved. Images were generated using CytExpert, and data were processed using GraphPad Prism 10 software.

[0065] 3. Experimental Results The experimental results of treating the AML cell line MOLM13 with different concentrations of the ALK inhibitors brigatinib (BRI) and venetoclax (VEN), cytarabine (CYT), and daunorubicin (DAU) alone or in combination for 48 h are as follows: Figure 1As shown, the results indicated that the combination of brigatinib with veneclax, cytarabine, or daunorubicin significantly increased the apoptosis rate of AML cells. Furthermore, the CI < 1 for different concentrations of brigatinib combined with different concentrations of veneclax, cytarabine, or daunorubicin. This suggests that the concentration ratios of brigatinib with veneclax (0.625 μM:1.25 μM, 0.625 μM:2.5 μM, 1.25 μM:1.25 μM, 1.25 μM:2.5 μM), brigatinib with cytarabine (0.625 μM:0.625 μM, 0.625 μM:1.25 μM, 1.25 μM:0.625 μM, 1.25 μM:1.25 μM), and brigatinib with daunorubicin (0.625 μM:0.0125 μM, ... The combined use of brigatinib (0.025 μM, 0.0125 μM, and 0.025 μM) in the treatment of leukemia showed significant synergistic effects. These results indicate that brigatinib, in combination with verneclade, cytarabine, or daunorubicin, can synergistically induce apoptosis in AML cells and possesses synergistic anti-AML cell activity. Furthermore, brigatinib can induce apoptosis in chronic myeloid leukemia cells (KU812), T-cell acute lymphoblastic leukemia cells (CCRF-CEM), or B-cell acute lymphoblastic leukemia cells (Sup-B15), demonstrating activity against chronic myeloid leukemia, T-cell acute lymphoblastic leukemia, and B-cell acute lymphoblastic leukemia.

[0066] Example 2: Killing effect of brigatinib on leukemia cells 1. Experimental materials Leukemia cell lines: Kasumi-1, SKNO-1, MOLM-13, NOMO-1, HL60, THP-1, MV4-11, NB4, U937 and OCI-AML5; CML cell line: KU812; T-ALL cell lines: CCRF-CEM, HPB; B-ALL cell line: SUP-B15.

[0067] 2. Experimental Methods CCK-8 assay for brigatinib's IC50 in killing leukemia cell lines 50 The specific experimental steps are as follows: Brigatinib was serially diluted 2-fold from 20 μM using complete culture medium. Four replicates were then set up for each concentration, with 50 μL of each concentration added to the corresponding well of a 96-well plate. 100 μL of the corresponding complete culture medium was added to the control wells. 50 μL of cell suspension was added to each drug-treated well, and the cells were incubated at 37°C. After 72 h, 10 μL of CCK-8 was added to each well for 4 h of incubation. The absorbance was then measured, and the data were saved and processed using GraphPad Prism software.

[0068] 3. Experimental Results Different concentrations of the ALK-selective inhibitor brigatinib were used to treat AML cell lines Kasumi-1, SKNO-1, MOLM-13, NOMO-1, HL-60, THP-1, MV4-11, NB4, U937, and OCI-AML5, T-ALL cell lines CCRF-CEM and HPB, CML cell line KU812, and B-ALL cell line Sup-B15 for 72 h. The viable cell percentage was detected using the CCK-8 assay, and the IC50 was calculated using GraphPadPrism. 50 Value, result as Figure 2 As shown in the results, different concentrations of the ALK selective inhibitor brigatinib can effectively kill various types of leukemia cell lines.

Claims

1. Use of brigatinib or a combination thereof with venetoclax, cytarabine or daunorubicin in the preparation of a medicament for preventing and / or treating leukemia.

2. Use according to claim 1, characterized in that, The use comprises the use of any one of the following medicaments in the preparation of a medicament for preventing and / or treating leukemia: (1) brigatinib or a pharmaceutically acceptable salt thereof; (2) brigatinib or a pharmaceutically acceptable salt thereof in combination with venetoclax or a pharmaceutically acceptable salt thereof; (3) brigatinib or a pharmaceutically acceptable salt thereof in combination with cytarabine or a pharmaceutically acceptable salt thereof; (4) brigatinib or a pharmaceutically acceptable salt thereof in combination with daunorubicin or a pharmaceutically acceptable salt thereof; optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the venetoclax or a pharmaceutically acceptable salt thereof is 1:1-1:8; optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the cytarabine or a pharmaceutically acceptable salt thereof is 1:0.1-1:5; optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the daunorubicin or a pharmaceutically acceptable salt thereof is 25:1-100:1; optionally, the leukemia is acute myeloid leukemia, chronic myeloid leukemia, T acute lymphoblastic leukemia or B acute lymphoblastic leukemia.

3. Use according to claim 1, characterized in that, The brigatinib or a combination thereof with venetoclax, cytarabine or daunorubicin significantly inhibits the proliferation of leukemia cells and / or promotes the apoptosis of leukemia cells.

4. Use according to claim 1, characterized in that, The medicament comprises an effective amount of brigatinib, an effective amount of brigatinib and venetoclax, an effective amount of brigatinib and cytarabine, or an effective amount of brigatinib and daunorubicin.

5. The use according to claim 1, characterized in that, The medicament further comprises a pharmaceutically acceptable excipient; optionally, the pharmaceutically acceptable excipient comprises a diluent, a binder, a disintegrant, a lubricant, a suspending agent, a stabilizer, a preservative and / or an osmotic pressure regulator; optionally, the dosage form of the medicament is an oral preparation, an injection or an intravenous drip; optionally, the oral preparation is a tablet, a capsule, a granule, a suspension, a powder, a pill, an emulsion or a syrup; optionally, the injection is a lyophilized powder injection, an injection solution, an injection suspension, an injection emulsion or a liposome injection.

6. A pharmaceutical composition for preventing and / or treating leukemia, characterized by, The pharmaceutical composition comprises any one of the following medicaments: (1) brigatinib or a pharmaceutically acceptable salt thereof; (2) brigatinib or a pharmaceutically acceptable salt thereof in combination with venetoclax or a pharmaceutically acceptable salt thereof; (3) brigatinib or a pharmaceutically acceptable salt thereof in combination with cytarabine or a pharmaceutically acceptable salt thereof; (4) brigatinib or a pharmaceutically acceptable salt thereof in combination with daunorubicin or a pharmaceutically acceptable salt thereof; optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the venetoclax or a pharmaceutically acceptable salt thereof is 1:1-1:8; optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the cytarabine or a pharmaceutically acceptable salt thereof is 1:0.1-1:5; optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the daunorubicin or a pharmaceutically acceptable salt thereof is 25:1-100:

1.

7. A pharmaceutical preparation for preventing and / or treating leukemia, characterized by, The pharmaceutical preparation comprises the pharmaceutical composition of claim 6.

8. The pharmaceutical preparation according to claim 7, characterized in that The dosage form of the pharmaceutical preparation is an oral preparation, an injection or an intravenous drip; optionally, the oral preparation is a tablet, a capsule, a granule, a suspension, a powder, a pill, an emulsion or a syrup; Optionally, the injection is a lyophilized powder injection, an injection solution, an injection suspension, an injection emulsion or a liposome injection.

9. A method of inhibiting proliferation and / or promoting apoptosis of leukemia cells in vitro for non-therapeutic purposes, characterized in that, The method comprises treating leukemia cells with any one of the following drugs: (1) brigatinib or a pharmaceutically acceptable salt thereof; (2) brigatinib or a pharmaceutically acceptable salt thereof in combination with venetoclax or a pharmaceutically acceptable salt thereof; (3) brigatinib or a pharmaceutically acceptable salt thereof in combination with cytarabine or a pharmaceutically acceptable salt thereof; (4) brigatinib or a pharmaceutically acceptable salt thereof in combination with daunorubicin or a pharmaceutically acceptable salt thereof. Optionally, the leukemia cells are Kasumi-1, SKNO-1, MOLM-13, NOMO-1, HL-60, THP-1, MV4-11, NB4, U937, OCI-AML5, CCRF-CEM, HPB, KU812 or Sup-B15. Optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the venetoclax or a pharmaceutically acceptable salt thereof is 1:1-1:8; Optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the cytarabine or a pharmaceutically acceptable salt thereof is 1:0.1-1:5; Optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the daunorubicin or a pharmaceutically acceptable salt thereof is 25:1-100:

1. Optionally, the leukemia is acute myeloid leukemia, chronic myeloid leukemia, T acute lymphoblastic leukemia or B acute lymphoblastic leukemia.

10. The use of any one of the following aspects: (1) brigatinib or a combination thereof with venetoclax, cytarabine or daunorubicin in the preparation of a pharmaceutical preparation for preventing and / or treating leukemia; (2) brigatinib or a combination thereof with venetoclax, cytarabine or daunorubicin in the preparation of a drug for inhibiting the proliferation of leukemia cells and / or promoting the apoptosis of leukemia cells in vitro for non-therapeutic purposes. Optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the venetoclax or a pharmaceutically acceptable salt thereof is 1:1-1:8; Optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the cytarabine or a pharmaceutically acceptable salt thereof is 1:0.1-1:5; Optionally, the mass ratio of the brigatinib or a pharmaceutically acceptable salt thereof to the daunorubicin or a pharmaceutically acceptable salt thereof is 25:1-100:

1. Optionally, the leukemia is acute myeloid leukemia, chronic myeloid leukemia, T acute lymphoblastic leukemia or B acute lymphoblastic leukemia.

Images